Bloodstream infections (BSI) are among the most severe manifestations of bacterial disease. The detection of bacteria in blood has an important role in the diagnosis of the patient, and blood culture is still considered to be the reference method of diagnosis in a clinically suspected case of BSI.
Development of a rapid diagnostic test for detecting bacterial infection in blood or any other tissue would have a significant impact on the management of infections. For the identification of pathogens and antibiotic resistance genes in clinical samples, DNA probe and DNA amplification technologies offer several advantages over conventional methods. The organism can be detected directly in clinical samples, in donated or pooled blood, in biopsy or autopsy samples, or tissue or organs donated for transplant, thereby reducing the cost and time associated with isolation of pathogens. Also, bacterial genotypes (at the DNA level) are more stable than the bacterial phenotypes (i.e. biochemical properties). DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory (and a method to quantify small amounts of DNA). For example, kits for the detection of fastidious organisms based on the use of hybridization probes or DNA amplification for the direct detection of pathogens in clinical specimens are commercially available.
DNA-based tests for detection and identification of bacteria could be based on the amplification of the highly conserved 16S rRNA gene followed by hybridization with internal species-specific oligonucleotides. The significance of the 16S rRNA genes is that certain sequences are conserved in virtually all species. The subsequent hybridization targets allow for amplification of species-specific oligonucleotides which are derived from species-specific bacterial genomic DNA fragments. However, ultimately, these straightforward strategies using broad-based “universal” sequences suffer from the fact that the use of normal Taq polymerase (which is contaminated with bacterial nucleic acid(s)) interferes with the detection. Contamination of the Taq polymerase with bacterial nucleic acid was first described over 20 years ago. See Rand and Houck, Molecular and Cellular Probes (1990) 4:445-450. This means if one uses primers targeting areas of the 16 S ribosomal RNA (or DNA) that are shared by many bacteria, the contamination of the Taq with these sequences becomes a limiting factor in detecting low copy numbers of bacteria. In applying such a method to the detection of bacteria in normally sterile clinical specimens, Taq enzyme contamination forces the use of primers specific to various species of bacteria, rather than allowing the use of sequences that could amplify all or many species.